In all eukaryotic cells, entry into mitosis is controlled by the Cyclin-dependent kinase, Cdc2. Cdc2/Cyclin B complexes are controlled by the inhibitory kinases, Myt1 and Wee1 and by the activating phosphatase, Cdc25. Although a number of Cdc25 and Myt1/Wee1-regulatory pathways have been identified, how these pathways are interconnected and which upstream modulators impinge on these pathways is not yet entirely clear. To understand how physiological stimuli and checkpoint control pathways regulate the G2/M transition, it is essential that we understand the causal connections and regulatory interplays between these diverse regulators. This proposal employs the powerful in vitro reconstitution system for mitotic entry provided by Xenopus egg extracts to investigate Cdc25 and Wee1 regulation. In this proposal we address several outstanding issues in G21M regulation, including the mechanism by which Cdc25 is activated at mitosis through removal of bound 14-3-3 proteins, whether Cdc25-directed activating phosphatases may be cell cycle regulated and how different isoforms of Cdc25 may be differentially regulated. We also investigate several candidate regulators of Wee1, including the kinase PIx and the methyltransferase, Hsl7. Our long-term objective is to obtain a mechanistic understanding of the molecular events that control entry into mitosis.